U.S. patent application number 09/791105 was filed with the patent office on 2002-02-21 for method for detecting the presence of at least one single allele of a deletion mutant.
Invention is credited to Brinkmann, Ulrich, Kerb, Reinhold, Schlagenhaufer, Robert, Sprenger, Raimund.
Application Number | 20020022225 09/791105 |
Document ID | / |
Family ID | 8167946 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022225 |
Kind Code |
A1 |
Sprenger, Raimund ; et
al. |
February 21, 2002 |
Method for detecting the presence of at least one single allele of
a deletion mutant
Abstract
Method for detecting the presence of at least one single allele
of a deletion mutant, specially as PCR assay for detecting the
presence of at least one GST1*0 allele wherein a PCR is performed
with two primers, of which one stems from the sequence upstream of
the deletion area, and the other stems from the sequence downstream
of the deletion area and wherein the production of the
corresponding DNA fragment in the PCR is checked. Useful for
testing of patients to check whether they are susceptible to toxins
or resistant or overly sensitive to certain therapeutic agents or
belonging to risk groups.
Inventors: |
Sprenger, Raimund;
(Weilheim, DE) ; Schlagenhaufer, Robert;
(Starnberg, DE) ; Brinkmann, Ulrich; (Bernried,
DE) ; Kerb, Reinhold; (Munchen, DE) |
Correspondence
Address: |
C. Dean Domingue of Domingue & Waddell, PLC
First National Bank Towers, Suite 515
Box 75
600 Jefferson Street
Lafayette
LA
70501
US
|
Family ID: |
8167946 |
Appl. No.: |
09/791105 |
Filed: |
February 22, 2001 |
Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6886 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 24, 2000 |
EP |
00103844.7 |
Claims
1. Method for detecting the presence of at least one single allele
of a deletion mutant, characterized in that a PCR is performed with
two primers, of which one stems from the sequence upstream of the
deletion area, and the other stems from the sequence downstream of
the deletion area, wherein the production of the corresponding
DNA-fragment in the PCR is checked.
2. PCR-Assay for detecting the presence of at least one GSTT1*0
allele characterized by using a combination of one primer from the
enclosed sequence 1 and one primer from the enclosed sequence 2 and
obtaining specific DNA-fragments for this allele by PCR.
3. Assay according to claim 1, characterized by using the
combination of the primers CAG TTG TGA GCC ACC GTA CCC and CGA TAG
TTG CTG GCC CCC TC
4. Procedure for diagnostic testing of individuals to check whether
they are susceptible to toxins or resistant to certain therapeutic
agents or belonging to risk groups characterized by obtaining blood
samples from the individuals and preparing genomic DNA from these
blood samples PCR mapping of the obtained DNA using a combination
of one primer from the enclosed sequence 1 and one primer from the
enclosed sequence 2 and analysing whether corresponding DNA
fragments have been produced by PCR.
5. Procedure according to claim 4, characterized by further PCR
mapping of the obtained DNA using a primer pair from within the
GSTT1 gene and analysing whether PCR fragments of the primers
according to claim 3 and/or PCR fragments of the primers from
within the GSTT1 gene have been produced.
6. Procedure according to claim 4 or 5, characterized in that the
risk for UV-mediated skin damage and/or the genetic risk for skin
cancer and/or the genetic risk for cancers that are associated with
oxidative stress and/or damage is predicted according to the
results of the PCR mapping.
7. Quantitative PCR assay for detecting the number of active GSTT1
alleles using the ABI TaqMan.RTM. technology.
8. Use of the knowledge of exact GSTT1 copy numbers obtained
according to claim 1 to develop quantitative PCR based assays for
detecting the number of active GSTT1 alleles using the ABI
TaqMan.RTM. technology.
9. Use of the knowledge of exact GSTT1 copy numbers obtainted
according to claim 1 to calibrate quantitative PCR based assays for
detecting the number of active GSTT1 alleles using the ABI
TaqMan.RTM. technology.
10. Use of the knowledge of exact GSTT1 copy numbers obtained
according to claim 1 to calibrate assays for detecting the number
of active GSTT1 alleles.
Description
[0001] This invention relates to a method for detecting the
presence of at least one single allele of a deletion mutant, a PCR
assay for detecting the presence of at least one GSTT1* allele and
a procedure for diagnostic testing of patients to check whether
they are susceptible to toxins or resistant to certain therapeutic
agents or belonging to risk groups.
[0002] Human glutathione S-transferase theta (GSTT1) is an
important detoxification enzyme comprising a deletion polymorphism.
Approximately 20% of Caucasians are homozygous GSTT1*0/0 failing to
express any GSTT1 activity. Non conjugators may have an impaired
ability to metabolically eliminate toxic compounds and may
therefore be at increased risk for cancer, inflammatory diseases or
chemical poisoning.
[0003] Any conclusion drawn from current genotyping was limited
because heterozygous (*A/0) and homozygous (*A/A) samples could yet
not be discriminated. Phenotypically suggested high- and
intermediate conjugators remained genotypically unexplained. The
classification of all three genotypes has so far been hampered by
the elucidation of the correct molecular mechanism of the GSTT1
deletion.
[0004] Thus, it is the object of this invention to provide a method
for detecting the presence of at least one single allele of this
deletion mutant.
[0005] This problem is solved by a method for detecting the
presence of at least one single allele of a deletion mutant,
wherein a PCR is performed with two primers, of which one stems
from the sequence upstream of the deletion area, and the other
stems from the sequence downstream of the deletion area, wherein
the production of the corresponding DNA fragment in a PCR is
checked.
[0006] It is preferred to use this invention for detecting the
presence of at least one GSTT1*0 allele, wherein a combination of
one primer form the enclosed sequence 1 and one primer from the
enclosed sequence 2 is used and specific DNA fragments for the
GSTT1*0 allele are obtained by PCR.
[0007] Thereby, using the following primers showed very good
results:
1 CAG TTG TGA GCC ACC GTA CCC CGA TAG TTG CTG GCC CCC TC
[0008] A special purpose of this invention is to use the invention
in a procedure for diagnostic testing of patients to check whether
they are susceptible to toxins or resistant to certain therapeutic
agents or belonging to risk groups, wherein
[0009] blood samples from the patients are obtained and genomic DNA
is prepared from these blood samples
[0010] a PCR-Mapping of the obtained DNA is performed using a
combination of one primer from the enclosed sequence 1 and one
primer from the enclosed sequence 2 and
[0011] it is analysed whether corresponding DNA-fragments have been
produced by the PCR.
[0012] In a preferred embodiment, there is an additional PCR
mapping of the obtained DNA using a primer pair from within the
GSTT1 gene and it is analysed whether PCR fragments of the primers
according to claim 3 and/or PCR fragments of the primers from
within the GSTT1 gene have been produced. In this way, all possible
allele combinations (GSTT1*0/0, GSTT1*A/0 and GSTT1*A/A) can be
detected.
[0013] It is preferred to use this GSTT1-genotyping assay to
predict the risk for UV mediated skin damage and/or the genetic
risk for skin cancer and/or the genetic risk for cancers that are
associated with oxidative stress and/or damage.
[0014] According to this invention, it is further possible to
obatin a quantitative PCR assay for detecting the number of active
GSTT1 alleles using the ABI TaqMan.RTM. technology.
[0015] With the method according to this invention, it is
preferrably possible to use the knowledge of the exact GSTT1 copy
number to develop quantitative PCR based assays for detecting the
number of active GSTT1 alleles using the ABI TaqMan.RTM.
technology.
[0016] It is further possible according to the invention to use the
knowledge of the exact numbers of GSTT1 copoies obtained according
to the invention to calibrate quantitative PCR based assays for
detecting the number of active GSTT1 alleles using the ABI
TaqMan.RTM. technology.
[0017] The knowledge of the exact GSTT1 copy numbers obtained
according to the invention can also be used to calibtrate assays
for detecting the number of active GSTT1 alleles.
[0018] In the following, the invention is explained in detail with
reference to the enclosed figures and tables, wherein
[0019] FIG. 1 shows the structure of the GSTT1 gene region and
mapping of the deletion and the genomic organisation of GSTT1 (*A
allele)
[0020] FIG. 2 shows the mapping of the deletion and primer
localization as well as selective primer combinations and
corresponding PCR fragments for mapping of the deletion:
[0021] Primer Combinations:
2 Primer Combinations: lane 1 and 2: GST-TF13/TRF13, lane 3 and 4:
GST-TF12/TRF12, lane 5 and 6: GST-TR11/TFR11, lane 7 and 8:
GST-TR13/GST-TRF13;
[0022] FIG. 3 shows the differentiation of GSTT1 genotypes by PCR
assays:
[0023] a) Method according to the invention: a 1,5 kb fragment
defines the *0/0 genotype, a 1,5 kb and 466 bp fragment indicate
*A/O and a 466 bp fragment *A/A.
[0024] b) Method according to the state of the art: no fragment
defines the *0/0 genotype, a 459 bp fragment indicates *A/0 or
*A/A;
[0025] wherein lanes 1-3 represent samples from completely deleted
(*0/0) individuals, lanes 4-6 from heterozygous (*A/0) and lanes
7-9 from homozygous *A individuals (*A/A), a 100 bp ladder (M) was
used; identical samples were investigated;
[0026] FIG. 4 shows the sequence and the mechanism of the GSTT1
deletion:
[0027] The schematic representation of the recombination event
above and the sequence of the recombinant fragment;
[0028] FIG. 5 shows the frequency distribution of GSTT1 conjugation
activity in 130 samples;
[0029] FIG. 6 shows the mean UV skin sensivity by minimal erythema
dose according to GSTT1 genotype in 60 healthy volunteers;
[0030] FIG. 7 shows the ratio between GSTT1 and CCR 5 in dependance
on the number of PCR cycles;
[0031] FIG. 8 shows the clusters with low, intermediate and high
flurescense ratios corresponding to the genotypes GSTT1*0/0,
GSTT1*A/0 and GSTT1*A/A respectively;
[0032] Table 1 shows the primers for the characterization of the
GSTT1 deletion locus and diagnostic PCR;
[0033] Table 2 shows the GSTT1 allele distribution and phenotype
correlation;
[0034] Table 3 shows the PCR fragments for GSTT1 genotyping;
[0035] Table 4 shows the skin sensitivity to UV irradiation
dependent on GSTT1 genotype; and
[0036] Table 5 shows cluster analysis and statistical significance
of the genotype correlation.
[0037] The invention characterizes the structure and mechanism of
the GSTT1 deletion by PCR mapping and sequencing: a 54251 bp
fragment carrying the GSTT1 gene was deleted from the functional
allele by a homologous recombination event. The deletion
breakpoints are concealed within a 403 bp region on the null
allele.
[0038] Based on this data a PCR assay using primer pairs
(CAGTTGTGAGCCACCGTACCC, CGATAGTTGCTGGCCCCCTC) and
(CCAGCTCACCGGATCATGGCCA- G, CCTTCCTTACTGGTCCTCACATCTC) for GSTT1*0
and GSTT1*A, respectively, has been established according to the
invention, that revealed all three GSTT1 genotypes (GSTT1*0/0,
GSTT1*A/A, and GSTT1*A/0).
[0039] Furthermore, a Mendelian intermediary inheritance was proved
by correlating the GSTT1 genotype with the enzyme activity using
the substrate dichloromethane. Samples with two active alleles
(GSTT1*A/A) expressed a statistically significant higher enzymatic
activity compared to those with one null allele (p<0.0001,
ANOVA).
[0040] This improved method can be introduced into routine
genotyping as a new diagnostic tool and will help to elucidate the
clinical relevance of this gene.
[0041] Glutathione S-transferase theta enzyme activity involved in
the metabolism of toxic compounds is absent in approximately 20% of
Caucasians due to a homozygous deletion of GSTT1 (*0/0). Because
the exact manner of the GSTT1 deletion was unknown, current
genotyping of GSTT1 was limited to detect the presence vs complete
absence of the gene by a GSTT1-specific PCR. Thus, heterozygous
(*A/0) and homozygous (*A/A) samples could not be discriminated.
The invention characterizes the boundaries of the deletion of the
human glutathione S-transferase theta (GSTT1) gene: PCR mapping and
sequencing revealed a 54251 bp fragment including GSTT1 to be
deleted from chromosome 22, most likely by a homologous
recombination event between two highly homologous sequence
stretches that flank GSTT1. Based on the knowledge of the GSTT1*0
region, a PCR assay was devised for unambiguous discrimination of
homozygously deleted (*0/0), heterozygously (*A/0) and homozygously
GSTT1 carrying (*A/A) individuals. Genotyping of 180 samples of a
Caucasian population revealed that the deletion consists of one
defined allele, whose distribution in the population fits the
Hardy-Weinberg equilibrium with observed 20% *0/0, 46% *A/0 and 34%
*A/A individuals. The number of GSTT1*A alleles detected by this
procedure correlated highly significant with the enzyme activity in
erythrocytes. Genotype-phenotype comparisons proved a codominant
type of inheritance by a gene-dose effect: samples with two active
alleles expressed a statistically significant higher enzymatic
activity compared to those with one null allele (p<0.0001,
ANOVA).
[0042] Glutathione S-transferases (GSTs), a multi gene family of
enzymes comprising several classes (class alpha, mu, pi, theta, and
zeta), are expressed in many tissues, including liver, lung, heart,
intestine, erythrocytes, and lymphocytes (Hayes et al., 1995). GSTs
regulate the conjugation of toxic compounds to excretable
hydrophilic metabolites. Because of that, their activity may affect
individual susceptibility to environmental toxins, carcinogens,
cancer, and other diseases (Strange 1999). The glutathione
S-transferase theta (GSTT1) gene, and its corresponding enzyme
activity is lacking in about 20% of Caucasians (Brockmoller et al.,
1996). However, even within the Caucasian population, this
frequency differs with respect to ethnicity (Nelson et al., 1995).
A nonfunctional GSTT1 allele (GSTT1*0) is the result of a partial
or complete deletion of the gene, the enzyme is completely absent
in homozygots (GSTT1*0/0) (Pemble et al., 1994). Many chemicals,
such as halogenated alkanes and epoxides whose use is widely spread
in industry, are substrates for GSTT1 and thus their toxicity can
be modulated by GSTT1. Interestingly, glutathione conjugation may
cause detoxification as well as toxification. For instance, the
conjugation of dichloromethane yields the toxic metabolite
formaldehyde (Hallier et al., 1994) and the mutagenicity of several
halogenated alkanes was enhanced in a GSTT1-expressing model system
(Their et al., 1996). Thus, GSTT1 polymorphism may determine
individual susceptibility towards toxic compounds. For example, of
two workers that were accidentally exposed to methyl bromide, the
GSTT1 conjugator suffered severe poisoning while the deficient
developed only mild neurotoxic symptoms (Garnier et al., 1996).
Since glutathione conjugation may provide a step in elimination of
substances which are toxic per se, GSTT1 activity can also act
protective (Ketterer et al., 1993). This explains that GSTT1
protected human lymphocytes from DNA and chromosomal damage after
exposure to several halomethanes (Wiencke et al., 1995; Hallier et
al., 1993). Consequently, GSTT1 has been investigated as risk
factor in epidemiological studies. The deletion conferred an
increased risk in myelodysplastic syndrome (Chen et al., 1996) and
some studies have suggested a correlation with susceptibility of
the skin to UV-irradiation (Kerb et al., 1997). Although
substantial data on epidemiological associations between GSTT1
deficiency and cancer exist, the results are conflicting, which
might be partly due to limitations of the currently used genotyping
procedures for GSTT1. Using biochemical analyses, three groups,
high-, intermediate-, and non-conjugators, can be discriminated
(Hallier et al., 1990). This may suggest a Mendelian intermediary
inheritance (Wiebel et al., 1999), but so far the genetic
background of intermediate and high conjugators and a gene-dose
effect could not be unambiguously established. The currently used
genetic assay for GSTT1 deficiency is a gene-specific PCR fragment
that is present in conjugators, and absent in GSTT1*0/0. The
fragment is therefore diagnostic for the presence of at least one
functional allele (GSTT1*A) and a differentiation between homo- and
heterozygous carriers of GSTT1*A is not possible, according to the
state of the art.
[0043] This invention shows the characterization of the GSTT1
deletion, probably the result of a recombination event between two
highly homologous regions that flank the GSTT1 gene. Utilizing the
sequence of the GSTT1*0 recombination region, a PCR assay was
devised that permits not only the unequivocal determination of
homozygously deleted (*0/0) but also the discrimination of the
heterozygously (*A/0) from the homozygously active (*A/A)
individuals. The three GSTT1 genotypes detected by this procedure
correlated highly significant with enzyme activity in erythrocytes.
The trimodular distribution of phenotypes with high-,
intermediate-, and null activity in homo- and heterozygotes for the
*A allele and *0/0 homozygotes, respectively, indicates a gene-dose
effect.
[0044] For initial determination and characterization of the GSTT1
deletion, samples of Caucasian volunteers from the Dr. Margarete
Fischer-Bosch-Institute of Clinical Pharmacology in Stuttgart, and
from the Institute of Clinical Pharmacology at the University
Medical Center, Charit in Berlin have been used. Samples were
obtained under consideration of all ethical and legal requirements.
Genomic DNA was prepared from blood using the Qiagen (QiaAmp) kits
on a Qiagen 9604 robot. For geno-phenotype correlations, phenotyped
subjects have been used (n=130, male, mean age 30.7 years, ranging
from 22 to 49 years) which were part of a previous study (Bruhn et
al. 1998), DNA was obtained from these samples using
phenol/chloroform extraction.
[0045] Determination of formaldehyde production rate (pmol
HCHO/min/.mu.l) from 31, 62, and 124 mM dichloromethane in
hemolysate was used as a measure for GSTT1 activity (Bruhn et al.,
1998).
[0046] NCBI database entries Z84718.1 and AP000351.2 (Genbank)
contain GSTT1 sequences in annotated form (Z84718.1) or as raw data
files, respectively. DNA sequence comparisons, alignments and the
construction of composite files from raw data sequence files were
performed using the programs FASTA and BLAST at the NCBI
server.
[0047] Specific oligonucleotide primers for PCR of GSTT1 gene
fragments from genomic DNA were derived (Table 1). Sequences of
purified PCR fragments were obtained by automated DNA sequencing on
ABI 377 (gel) or ABI 3700 (capillary) sequencers using BigDye
Terminator cycle sequencing reactions (PE Biosystems).
Amplification of fragments less than 2 kb was performed in 25 .mu.l
volume: 100 ng DNA template added to buffer containing 1.5 mM
MgCl.sub.2, 200 .mu.M dNTPs, 0.2 mM each primer and 1 U HotStarTaq
polymerase (all reagents Qiagen, Hilden, Germany). PCR was carried
out in a Perkin Elmer GeneAmp System 9700 with an initial
denaturation of 15 min at 95.degree. C. followed by 30 cycles of
94.degree. C. for 30 s, 30 s annealing and 60 s of extension at
72.degree. C. Final extension was carried out for 7 min at
72.degree. C. For longer amplicons 50 .mu.l PCR reactions contain
200 ng of genomic DNA, reaction buffer 3, 500 .mu.M dNTPs, 2.6 U
Expand Taq-System (Roche, Basel, Switzerland) and 0.3 mM primers
(Metabion, Munich, Germany). Samples were incubated at 92.degree.
C. for 2 min, followed by 35 cycles at 92.degree. C. for 10 s, 45 s
annealing at 68.degree. C. for each kb per min extension time. The
extension time of each cycle was increased by 20 s for the last
cycles. 10 min final extension at 68.degree. C. were applied.
[0048] The human GSTT1 gene is located on chromosome 22q11.2 which
has recently been completely sequenced in the course of the human
genome sequencing project (Dunham et al., 1999); prior to that,
partial chromosome sequences were available in public databases
from the Sanger Center. A 76799 bp DNA sequence of a BAC clone (gb:
Z84718.1) contains the GSTT1 and the GSTT2 gene. Homology searches
in unnotated raw sequences revealed one additional GSTT1-containing
clone, AP000351.2 (118999 bp) consisting only of preliminary BAC
sequences which are not assembled to a defined linear gene. A
defined sequence file that contains GSTT1 and the flanking regions,
which extends the annotated GSTT1 gene region of clone Z84718.1 was
constructed by homology alignments using FASTA and BLAST at the
NCBI server. Due to the high homology of parts of the sequences,
the stringency of the alignments was set higher than default and
the correctness of the assembled sequence was confirmed by visual
inspection. The composition and prominent features of the GSTT1
gene region are shown in FIG. 1: GSTT1 and the homologous GSTT2
gene (55% protein homology, Tan et al., 1995) are separated by
49741 bp. In addition to many repetitive elements, GSTT1 is flanked
by 18-kb regions which are more than 90% homologous (defined in the
*A i.e. non-deleted allele, as homology region HA5 upstream and HA3
downstream of GSTT1).
[0049] Although the detailed site of the GSTT1 deletion was
unknown, the homozygous null allele could be diagnosed by the
absence of a PCR fragment that is specifically amplified from the
GSTT1 coding region (Pemble et al., 1994). The deletion removes
GSTT1 but not GSTT2 (Tan et al., 1995). Therefore, one breakpoint
of the deletion is positioned between GSTT1 and GSTT2 and the other
must be downstream of GSTT1 (FIG. 1). To map the GSTT1 deletion we
determined the presence or absence of specific sequences upstream
and downstream of GSTT1 by PCR. Ten samples of the genotype
GSTT1*0/0, and ten samples with at least one GSTT1*A allele were
preselected (see FIG. 3a). Primer sets for specific amplification
of fragments up- and downstream of GSTT1 were generated. This
required extensive optimization considering the highly homologous
flanking regions of GSTT1. Fragments that could be amplified from
both, GSTT1*0/0 and samples containing at least one *A allele (*A)
were regarded to be outside the deletion, whereas fragments that
could exclusively be amplified from *A samples, but not from *0/0
contain the region which is deleted. FIG. 2 shows results of this
mapping procedure. Primer combinations that bind upstream from
position 50191, and downstream from position 110007 of the sequence
file can be obtained from all samples. Fragments closer to GSTT1,
downstream position 53494 and upstream position 105675 cannot be
amplified from *0/0 samples. Therefore, the boundaries of the
deletion must be localized within a region between position 50192
to 53493 upstream and 105676 to 110006 downstream of GSTT1.
[0050] To home in on the exact positions of the deletion, we
applied long range PCR has been applied to span the deletion in
*0/0 samples. Various sets of PCR primers were selected close to
the deletion boundaries as defined by PCR-mapping. In GSTT1*A
samples, the 5' and 3' primers were separated by more than 60 kb,
in *0/0 samples the distance between these primers was reduced by
the size of the deletion. Utilizing different primer combinations,
reproducibly deletion-spanning PCR fragments of 10065 bp, 3187 bp
and of 1460 bp have been generated (FIG. 3, Table 3 for primer
positions). The 10 kb fragment was sequenced to characterize the
deletion region. The comparison of this null allele sequence with
the GSTT1*A allele revealed the boundaries of the deletion (FIG.
4): it is flanked upstream and downstream by sequences that are
part of the highly homologous HA5 and HA3 regions. In the *A
allele, these regions flank GSTT1. In *0, the deletion generates a
fusion sequence (H0) which differs from HA5 and HA3 by only a few
nucleotide deviations as shown in FIG. 4. Using these deviations as
"markers", for identification of the HA5 or HA3 portions of H0, the
region where the deletion had happened could be narrowed down to a
403 bp sequence stretch. This sequence is identical in HA5 and HA3.
These data support the assumption that the mechanism which
generated the deletion is homologous recombination between the
regions HA5 and HA3, which removes GSTT1 and generates H0.
[0051] The method according to the state of the art to analyze
GSTT1 includes a critical negative test output: lack of a PCR
signal defines *0/0 samples, whereas generation of the fragment
detects the presence of at least one GSTT1*A allele (*A/A or *A/0).
In addition to problems associated with negative test readouts
(e.g. false results due to test failure may be misinterpreted as
*0/0), another drawback of that method is that homozygous (*A/A)
and heterozygous (*A/0) samples cannot be distinguished. Utilizing
the molecular composition of the deletion, the invention devises a
new genotyping protocol that allows not only a positive detection
of the deletion allele, but also permits the unambiguous
discrimination of all GSTT1 genotypes (*A/A, *A/0, and *0/0). The
procedure generates deletion-spanning PCR fragments, which are
combined with fragments that indicate the presence of GSTT1.
Various PCR assays were evaluated using GSTT1 fragments in
combination with deletion spanning fragments of sizes between 10065
and 1460 bp (table 3). Among these, one assay that had been
established was found to be "robust" and allowed a reproducible
simultaneous discrimination of all genotypes. The 1460 bp
deletion-specific PCR fragment was combined with a 466 bp fragment
that detected GSTT1*A. FIG. 3 shows the results of this assay
applied to known *0/0 samples and *A/? samples: all samples that
had previously been genotyped homozygous for the deletion by the
standard method confirmed the *0/0 genotype with a positive readout
of 1460 bp. The genotype GSTT1*A/A is diagnosed by the single 466
bp fragment and can be differentiated from heterozygous individuals
who are characterized by the presence of both fragments. Testing
more than 50 GSTT1*0/0 samples, no discrepancies were detected
between the methods according to the state of the art and according
to the invention.
[0052] The GSTT1*0/0 genotype correlates with the non conjugator
phenotype. To evaluate whether intermediate and high conjugators
are caused by *A/0 and *A/A genotypes, 130 samples whose GSTT1
activity in blood had been determined have been genotyped. FIG. 5
shows that the three GSTT1 genotypes could be assigned to distinct
phenotypes: enzyme deficiency in GSTT1*0/0 samples, intermediate
activity in *A/0 samples, and high activity in *A/A samples has
been observed. The correlation of genotype and phenotype is
statistically highly significant, p<0.0001 for all group
comparisons (ANOVA with Bonferroni/Dunn correction for multiple
testing). The allelic frequencies were in agreement with
Hardy-Weinberg's law, the difference between observed and expected
results (calculation based on the frequency of *0/0) was not
significant (Table 2).
[0053] Characterization and Mechanism of the GSTT1 Deletion:
[0054] The invention allows to characterize the structure and
mechanism of the GSTT1 deletion and identify two 18 kb homology
regions flanking GSTT1 which are involved in the deletion (most
likely crossing over) event that produced the *0 allele. Extensive
sequence identity between both repeat regions in the *A allele, and
between these repeats and the corresponding region in the deletion
allele allowed to define the deletion boundaries within a 403 bp
region. Gene deletion by homologous unequal crossing over has been
described in other detoxification enzymes, in cytochrome P450 2D6
and glutathione S-transferase M1 (Steen et al., 1995; Xu et al.,
1998; Kerb et al., 1999). Like in GSTM1, the GSTT1 deletion has a
high frequency in the Caucasian population. Does this deletion
consist of one defined null allele or are there various deletions
with the loss of GSTT1 as the common denominator ? Since the assay
showed the deletion allele as "measurable" PCR fragment, any
variations in the size of the deletion indicative of multiple
deletion alleles would have been detectable. In more than 150 *0
allele harboring samples that have been analyzed, the "deletion
fragment" showed the same size of 1460 bp, suggesting that all
GSTT1 deficiencies are caused by one allele.
[0055] Improved genotyping assay and allele distribution in
Caucasians: So far, genotyping could detect the absence of GSTT1,
but provided neither information about the boundaries of the
deletion nor about the precise genotype. In spite of the extreme
homology, a few single nucleotide variations specific for the
recombinant region allow to create an assay for the detection of
the inactive allele by presence of a PCR-fragment. A single PCR
assay that detects this deletion-spanning PCR-fragment, combined
with a fragment that indicates the presence of GSTT1, allows the
unambiguous discrimination of all genotypes. Using the assay, the
allele distribution was analyzed in Caucasian individuals and found
to be 34% homozygous *A/A, 46% heterozygous, and 20% *0/0. This
frequency fits to the distribution that would be expected on the
basis of Hardy-Weinberg equilibrium (Table 2).
[0056] GSTT1 as risk factor in cancer: A number of epidemiological
studies have been published on the medical importance of GSTT1
(Strange and Fryer, 1999). GSTT1*0/0 was found to be associated
with brain cancer (Kelsey et al., 1997, Elexpuru Camiruaga et al.,
1995), head- and neck cancer (Cheng et al., 1999), lung cancer in
Hispanic- and African Americans (Kelsey et al., 1997). However,
results were often ambiguous (Duncan et al., 1995, Heagerty et al.,
1996) or gave conflicting results in bladder cancer (Kempkes et
al., 1996, Brockmoller et al., 1996) and colorectal cancer (Clapper
and Szarka, 1998; Zhang et al., 1999, Chenevix-Trench et al., 1995;
Katoh et al., 1995; Gertig et al., 1998). In all these studies only
a comparison of the null genotype with an active genotype was done.
The difference between heterozygous and homozygous active
individuals has not yet been elucidated, but determining both,
could improve the statistical power in epidemiological studies.
[0057] Genotype-phenotype Correlation:
[0058] Phenotypic data have indicated the presence of intermediate
conjugators displaying only half the activity of high conjugators
(Warholm et al., 1995). These observations, and a family study that
analyzed GSTT1 by semiquantitative PCR (Wiebel et al., 1999),
suggest a gene-dosage effect on GSTT1 activity. In this, it has
unambiguously been proven the intermediate Mendelian type of
inheritance of GSTT1 for the first time. The enzyme activity of
GSTT1 correlated highly significant with the number of functional
alleles and phenotypically classified intermediate- and high
conjugators were genotypically detected hetero (GSTT*A/0) and
homozygous (*A/A), respectively. In only 9 among a total of 130
samples the genotype did not correlate well with the phenotype.
Three intermediate conjugators had two active alleles and 6 samples
with high enzyme activity displayed unexpectedly only one active
allele. Since the enzymatic activity of all discrepant individuals
was close to the antimode and the enzyme assay had a CV of 7%,
differences are most likely the result of biological variability.
With codominant inheritance, each allele confers a measurable, yet
variable component to the phenotype resulting in a wide range of
enzymatic activities from a distinct genotype and overlapping
activities between homo- and heterozygotes. Furthermore, GSTT1
genotype-phenotype discrepancies can be modulated by exposure to
inducers or inhibitors, whereas the genotype remains constant. Two
other subjects attracted our attention because their extraordinary
high conjugation activity was 2-fold higher than the mean of
homozygous conjugators (68 pmol/min/.mu.l versus 32 pmol/min/.mu.l,
FIG. 5). These subjects displayed the *A/A genotype in our assay.
One possible explanation could be a gene duplication or
amplification of GSTT1. Members of the GST multigene family have
been evolutionary derived from a Theta-class gene duplication
(Pemble and Taylor, 1992), and a duplicated class M1 gene that
causes ultrarapid enzyme activity has already been described
(McLellan et al., 1997). Thus, it is feasible that rare (2 of 130)
ultrahigh GSTT1 activity may be caused by additionally amplified
gene copies.
[0059] The identification of GSTT1 genotypes with a procedure that
unambiguously discriminates *0/0, *A/0 and *A/A alleles predicts
highly significant the phenotype and will allow an accurate
assessment of health risk from halogenated alkanes or pesticides
(Bruning et al., 1997; Au et al., 1999; El-Masri et al., 1999). It
also provides a useful approach for the evaluation of the
importance of GSTT1 as risk factor for various diseases.
[0060] This invention is especially suitable to check whether an
individual has a genetic risk for UV mediated skin damage and/or
skin cancer and/or cancers that are associated with oxidative
stress and/or damage.
[0061] Ultraviolet (UV) irradiation by sun exposure and family
history are risk factors for the development of cutaneous melanoma.
Inherited susceptibility to this type of skin cancer could
therefore result from genetic factors that affect the capacity of
cells to prevent UV-induced DNA lesions. UV light mediates the
formation of radical oxygen species (ROS) such as hydroxyl and
superoxide radicals, hydrogen peroxide, and single oxygens. These
molecules comprise "oxidative stress" and damage cellular proteins,
lipids, and DNA. Oxidative stress can cause inflammation,
mutations, and genotoxicity. The skin is equipped with a defense
system against oxidative stress (Vessey, 1993). Glutathione
S-transferases (GSTs) contribute to this protection either by
direct inactivation of peroxidized lipids and DNA (Berhane et al.,
1994; Ketterer and Meyer, 1989; Tan et al., 1988), or by
detoxification of xenobiotics, which can serve as cofactors of
radical formation. Our improved assay system to detect GSTT1
deficiencies and heterozygotes shows that GST genotypes affect the
susceptibility of individuals to oxidative or chemical stress. FIG.
6 shows the results of a panel comparison of two groups of
genotypically characterized GSTT1 deficient, homozygous and
heterozygous active subjects in respect to sunlight (UV)
sensitivity. The GSTT1 genotyping was performed with the assay
according to the invention that is described above. Healthy
subjects (54 male, 6 female, 18 to 48 (mean 27.6) years old and of
German Caucasian origin were selected for the panel comparison
study. The constitutional skin types were assigned from tanning and
burning histories using the Fitzpatrick's Classification
(Fitzpatrick et al., 1987) and the study was performed during the
winter months. Reactivity to UV light was determined in eight skin
fields (1.times.0.6 cm) on non-UV exposed buttock skin by
increasing doses of simulated sunlight with 20% dose increments
(Wucherpfennig, 1931). Before each irradiation, UV intensity of the
radiation source (dermalight 2001% equipped with an h2 filter, Dr.
Hohnle, Munich, Germany) was calibrated to 1.33 mW/cm.sup.2 at
280-315 nm, by use of a UVA/B Meter (Dr. Hohnle). UV dose [J/cm2)
was calculated by intensity [mW/cm.sup.2].times.time [sec]/1000.
Dose variations, from 0.07 J/cm.sup.2 to 0.34 J/cm.sup.2, were
achieved by irradiation time. Erythema reactions were scored
visually 20 hours post-exposure, independently by two examiners.
All skin reactions were photographically documented. The
irradiation dose of the first field with a barely perceptible
erythema determined the minimal erythema dose (MED). The software
package for statistical analyses SPSS 10.0 was applied to evaluate
genotype-phenotype correlation and statistical significance (SPSS
Inc., Chicago, USA). The dependency of UV sensitivity from genotype
was tested with ANCOVA to control for constitutional skin type as
confounding variable. Adjustment for multiple testing was done
according to Bonferroni/Dunn.
[0062] The results of the panel comparison study showed a clear
correlation of GSTT1-genotype and UV sensitivity of the skin: The
GSTT1-deficient group has a higher inflammatory response after
exposure to UV irradiation compared to the group with GSTT1
activity. Subjects homozygous for the GSTT1 deletion mutation
required a statistically significant lower UV dose to barely
perceptible erythema than those with one (GSTT1*A/0; p=0.067) or
two (GSTT1*A/A; p=0.032) functionally active alleles. The erythema
reaction did not decrease with the number of functional alleles and
was equal in both GSTT1*A/0 and *A/A subjects. Among the 5 most
UV-sensitive subjects (MED.ltoreq.0.10 J/cm.sup.2) were 4 GSTT1
deficient and the two subjects with the lowest inflammatory
response (MED.gtoreq.0.29) were both of enzyme-expressing
genotype.
[0063] GSTT1 and GSTM1 are expressed in the skin and deficiencies
in these enzymes by gene deletions impairs the capacity of cells to
detoxify specific substrates, which include molecules that are
generated by oxidative damage. The modification of molecules with
reactive oxygens as substrate of GSTs provides one link between
GSTs enzyme activity and protection against UV radiation-induced
cutaneous damage. GST deficiencies result in increased
susceptibility of cells to the consequences of ROS attack, such as
inflammation or cancerogenesis (Fahey and Sundquist, 1991). UV
irradiation by sun exposure and family history are risk factors for
the development of skin cancer, particularly cutaneous melanoma.
The fact that the GSTT1 and, to a lesser degree the GSTM1 genotype
identifies individuals with increased UVsunlight-sensitivity,
suggests GSTT1 and -M1 genotype variations to be among the genetic
components that result in the inherited susceptibility or
predisposition to skin cancer.
[0064] This invention further relates to a method for detecting the
number of active alleles of the GSTT1 gene using the ABI
TaqMan.RTM. technology and to a method for calibrating assays to
detect the number of active GSTT1 alleles using quantitative
techniques (e.g. TaqMan, Light Cyler, MALDI-TOF). Based on the
molecular stucture of the GSTT1 gene a realtime quantitative
TaqMan.RTM. PCR assay was developed, that amplifies a fragment
within the coding sequence, and that coamplifies another fragment
outside the GSTT1 gene as internal standard, so that the ratio
between the yields of the both fragments (meassured by fluorescence
signals) in a certain PCR cycle is independent from factors like
template concentraion or DNA quality. Provided that the genomic
seuquence of the internal standard is non-polymorphic, the number
of active GSTT1 alleles is proportional to the described ratio. The
internal standard to be used here is based on a fragment within the
coding sequence of the CCR5 gene. Different polymorphic sites
according to CCR5 are known (32 bp Del, NT794, 1 bp Del, Arg223Gln,
Ala335Val, Cys303Ter), but none of them affect amplification of the
internal standard fragment. The quantitative GSTT1 Taqman Method,
described in this example, permits automized high throughput
analyses of individual GSTT1 genotype. Furthermore, genomic DNA of
lower quality than required for long range PCR (e.g. degraded DNA
as yielded from paraffin-embedded or formalin-fixated tissue
samples) can be used as template. The prerequisite for developing
the Taqman assay for GSTT1 genotyping is the knowledge of the exact
numbers of individual GSTT1 alleles as described in the previous
examples. Without knowing the exact number of the active GSTT1
alleles a calibration of the new assay is impossible. Therefore,
this method can be considered as a variation, or a direct
consequence, respectively, of the GSTT1 genotyping methods
described above.
[0065] The ABI TaqMan.RTM. technology bases on molecular probes
labelled by two fluorescence dyes: one is used as reporter and the
other is used as quencher. The probe is placed within the fragment
to be amplified. The 5'-3'-exonuclease activity of the Taq
polymerase hydrolyses the probe, so that the reporter dye is no
longer closed to the quencher dye and a fluorescence signal can be
measured after excitation at .lambda.=488 nm. The detected reporter
signal ist directly proportional to the amplified PCR fragment. For
coamplifying two fragments, two different reporter dyes must be
used.
[0066] An example for the successful application of quantitative
PCR (Taqman) to determine GSTT1 genotypes is shown below. The
reaction conditions for the assay, in which the CCR5 gene was used
as internal standard, were:
3 GSTT1 forward primer GTG CCC TTC CCT TAC CCA TC 88589-88570
reverse primer GGG TAC CAG TAG TCA GGG ACC TTA 88494-88517 probe
FAM-ACA GTG TGG CCA TCC TGC TCT ACC TGA-TAMRA 88554-88528 positions
according to GI: 9937243
[0067]
4 CCR5 forward primer TGG CCT GAA TAA TTG CAG TAG CT 804-826
reverse primer GTG CGT CAT CCC AAG AGT CTC T 879-858 probe VIC-TAA
GAG GTT GGA CCA AGC TAT GCA GGT GA-TAMRA 828-856 positions
according to GI: 2347111
[0068]
5 component amount template about 10 ng GSTT1 forward primer 0.4
.mu.M reverse primer 0.4 .mu.M probe 0.2 .mu.M CCR5 forward primer
0.2 .mu.M reverse primer 0.2 .mu.M probe TaqMan Universal PCR
Master Mix (ABI) 1 .times. water ad 25 .mu.L
[0069]
6 cycler ABI Prism 7700 reaction's volume 25 .mu.L initial steps
50.degree. C. 2 min 95.degree. C. 10 min denaturation 95.degree. C.
15 s annealing and extension 60.degree. C. 90 s cycle number 35
[0070] PCR data were exported as "clipped data" to be analysed, so
that for each well and each cycle one fluorescence signal value was
available. The ratios were built between the GSTT1 values and the
CCR5 values for each sample and each cycle and then were diagrammed
in dependance on the number of PCR cycles (FIG. 7).
[0071] The computerized determination of the GSTT1 copy numbers
from the output described in FIG. 7 was performed on data that were
obtained at cycle counts>cycle 25. The calculation of
fluorescence ratios from data obtained at cycles earlier than cycle
25 is not sensible, because the initial amplification's efficiency
is not the same for both fragments. Between cycles 25 and 30, the
amplicon yield is suifficiently high to calculate the ratio. In
later cycles the reaction kinetics of the PCR leads to a plateau
with no further increase of fluorescence signals, levelling
differences between the clusters and therefore making the
differentiation between homo- and heterozygous carriers of the null
allele impossible. Assay evaluation was performed from data
generated at 27 PCR cycles. Cluster analysis from the data output
of the quantitative PCR resulted in three distinctive clusters of
mean (min to max) signal ratios of -0.03 (-0.05--0.01), 0.28
(0.23-0.33), and 0.58 (0.48-0.54) (table 5). The three clusters did
not overlap and differed statistically highly significant from each
other (p<0.00001, ANOVA with Bonferroni-Dunn adjustment for
multiple testing) and the assignment to clusters was clear without
ambiguity. Calibrating this new Taqman assay by comparing the
results with those generated by the "method for detecting the
presence of at least one single allele of a deletion mutant"
revealed that the three clusters correlated completly with the
number of active GSTT1 alleles as indicated by a Spearman-Rho of
1.0 (p<0.001). FIG. 8 illustrates the clusters with low,
intermediate, and high fluorescence ratios corresponding to the
genotypes GSTT1*0/0, GSTT1*A/O, and GSTT1*A/A, respectively.
[0072] The results of this analysis, performed on multiple samples
of known GSTT1 genotype demonstrate the capability of the new
method to unambiguously determine the copy number of the GSTT1
allele in individual samples.
7TABLE 1 Primers for the characterization of the GSTT1 deletion
locus and diagnostic PCR Primer positions Name Primer sequences
[5'- . . . -3'] Annealing [.degree. C. ] 5' 3' Current Standard
Method GST-TF TTC CTT ACT GGT CCT CAC ATC TC 66 85920* 85898*
GST-TR TCA CCG GAT CAT GGC CAG CA 85462* 85481* PCR mapping primers
GST-TF13 CCC TCA CTC AGG GTT AGT GG 63 110007* 110026* GST-TRF13
GAT GCC ACG CGG CTT GTA GG 110301* 110282* GST-TF12 GAT TGG TGG AAG
GTG CCG GG 63 105553* 105576* GST-TRF12 CGT GTC TCT ACT TCA AAT TCC
ATG 105675* 105652* GST-TFR11 TAA GAT ACC TCA TAA AAT TAA CAG 59
53904* 53881* GST-TR11 GGG AGA ATG GAT AGT GGG GAG 53494* 53514*
GST-TFR13 GCA AGA AGA CCA GTG ACT GAG G 63 50191* 50170* GST-TR13
CTG CTC TTC TTC AGC AAC TCA G 49778* 49800* 10065 bp *0 fragment
primers GST-TRF13 GAT GCC ACG CGG CTT GTA GG 65 110301* 110282*
GST-TR12 CTT TTT CTG GAG CAA ACG CAT TG 45986* 46008* 3187 bp *0
fragment primers GST-TRF13.2 GAG CCA AGA AGT TCT GAG TCT TG 65
108037* 108015* GST-TR9n ATA TCA GCC AGA GAT CTC TGG G 50600*
50621* Sequencing primers GST-TRF13.3 GCA TCC CAA TTC AAC ACG TGT
TG 62 107075* 107053* GSTT-F.1000 CTT CTC AGC TGA AAC TTC CTC
51440* 51460* GSTT1 deletion assay primers GT*Af CCA GCT CAC CGG
ATC ATG GCC AG 70 85457* 85479* GT*Ar CCT TCC TTA CTG GTC CTC ACA
TCT C 85922* 85898* GT*0f GAG TTG TGA GCC ACG GTA CCC 52069*/6084**
52089*/6114** GT*0r CGA TAG TTG CTG GCC CCC TC 107779*/7543**
107760*/7524** *according to AP000351.2 **according to GSTT1*0
[0073]
8TABLE 2 GSTT1 allele distribution and phenotype correlation
Genotype *A/A *A/0 *0/0 N 44 60 26 % observed 33.8 46.2 20.0 %
expected.sup.a 30.6.sup.b 49.4.sup.b 20.0.sup.b Mean (SD) enzyme
activity 32.1(10.2).sup.c 15.0(7.4).sup.c 3.3(0.9).sup.c
.sup.abased on *0/0, according to Hardy-Weinberg .sup.b.chi..sup.2
= 0.23 .sup.cp < 0.0001, ANOVA with Bonferroni/Dunn correction
for multiple testing
[0074]
9TABLE 3 PCR-fragments for GSTT1 genotyping. Sequence Position Size
[bp] Specificity Comment GATGCCACGCGGCTTGTAGG 45986-46008 10065
GSTT1*0 Long range PCR CTTTTTCTGCACCAAACGCATTG 110301-110282
ATATCAGCCAGAGATCTCTGGG 50600-50621 3187 GSTT1*0 Long range PCR
CAGCCAAGAAGTTCTGAGTCTTG 108015-108037 CAGTTGTGAGCCACCGTACCC
52069-52089 1460 GSTT1*0 Standard PCR CGATAGTTGCTGGCCCCCTC
107779-107760 CCAGCTCACCGGATCATGGCCA 85457-85479 466 GSTT1*A
Standard PCR G CCTTCCTTACTGGTCCTCACATC 85922-85898 TC
[0075]
10 TABLE 4 GST-Enzyme MED (J/cm.sup.2).sup.a Activity n Mean Range
SD GSTT1*A/A High 15 0.164 0.10-0.24 0.043 GSTT1*A/0 Intermediate
22 0.157 0.12-0.29 0.048 GSTT1*0/0 Deficient 23 0.129 0.08-0.20
0.027 Total 60 0.145 0.08-0.29 0.037 .sup.aInflammatory reaction
was detected as minimal erythema dose (MED) at 20 h after
irradiation with UV
[0076]
11TABLE 5 Cluster analyses and statistical significance of the
genotype correlation Distance from Number Cluster cluster center 1
3 0.00 2 3 0.01 3 3 0.05 4 3 0.00 5 2 0.01 6 2 0.01 7 3 0.00 8 3
0.04 9 3 0.01 10 1 0.02 11 1 0.01 12 2 0.01 13 2 0.00 14 1 0.02 15
3 0.01 16 1 0.01 17 3 0.03 18 3 0.01 19 1 0.00 20 3 0.01 21 3 0.01
22 2 0.00 23 3 0.00 24 2 0.02 26 1 0.01 27 1 0.02 28 3 0.01 29 1
0.01 30 3 0.00 31 3 0.02 32 1 0.02 33 1 0.01 34 1 0.01 35 3 0.02 36
1 0.02 37 3 0.00 38 3 0.02 39 1 0.00 40 1 0.03 41 1 0.01 42 3 0.03
43 2 0.01 44 1 0.00 45 3 0.01 46 3 0.04 47 3 0.03 48 1 0.01 49 1
0.02 50 3 0.05 51 3 0.02 52 1 0.03 53 3 0.00 54 1 0.01 55 3 0.01 56
3 0.01 57 3 0.03 58 3 0.01 59 3 0.01 61 1 0.04 62 3 0.02 63 2 0.00
64 1 0.02 65 2 0.00 66 1 0.01 67 3 0.01 68 3 0.02 69 3 0.03 70 1
0.01 71 2 0.02 72 3 0.00 73 1 0.02 74 3 0.01 75 1 0.01 76 3 0.00 77
3 0.01 78 3 0.01 79 3 0.00 80 1 0.01 81 2 0.01 82 1 0.03 83 2 0.01
84 2 0.01 85 2 0.00 86 2 0.01 87 3 0.01 88 3 0.01 89 2 0.01 90 2
0.01 91 3 0.03 92 3 0.02 93 3 0.02 94 3 0.01 95 3 0.03 96 3
0.03
[0077]
Sequence CWU 1
1
2 1 5000 DNA Homo sapiens 1 gggtctggcg ggcaccagtg cgatggtgcg
ctgtacttgc ggcacaggta gtaaaggatg 60 gccgcgctgc agaaggggcc
ggtcaggggc actgcccttg ccttcctgag tgccactaca 120 tcaaccaccc
cggtgtggcc tgggcccaac tgctggggct tccagagcaa agaggagccc 180
aaacggcccc gagaaagacc ttcaccagag ctgtctgtct gacagtcagt aagggctggg
240 aaggagccct gcggggtgag taggagttgg gggctggtgg tataacaaag
agtaggccag 300 cagggggaac aacacgtgtt gaattgggat gctgaggtgg
gaggatcact tgatcccagg 360 aatttggggc tactgtgagc caagatcaca
ccactgcact ccagcttggg tgaaagatca 420 agatcctttt tcaaaaacaa
aaacgggggg gcacgatggc tcacacctgt aatcccggca 480 ctttgggagg
ccaatggggg cagatccctt gaggccagga gttggagacc agcctggcca 540
acatggtgaa accctgtctc tactaaaatg aaaatacaaa aattagctag ttgtggtggc
600 acacacctgt aatcccagct acttgggaag ctgaggcacg ggagtcactt
gaacctggga 660 ggcagaggtt gtagtgagcc aagattgtgc cactgtactc
cagcctgggc cacagagcaa 720 gactctgtct caaaaaacca acaaagaaaa
acacatgctg aaatacgagg gtaaagggag 780 caaggtaaat ctgaagaaaa
gagagtaggg ggttgcaact ggaagaaggg tgggggtgat 840 tggggagtga
tgaggcagcc agagacactg tggagtccac ggagggtagc ccctggaggt 900
gcagggaggt tatggactta atgcttaaga ttaggcatta tataagccag ggcatgaaag
960 gatccatctc tctggtgctg gatggagggt gagcccgagg gggcagaatg
gacaatgagg 1020 gggccagcaa ctatcgggaa ggttgtggtg tctgggaatg
ttggaggcca tggggacaga 1080 gggaagggga tggaggggag acatgcttcg
gaggggatgt cctaggcctt gctgattgat 1140 ggctggtgtg ggaacctccg
cagcacaagg gctcctttat catcaccagc agcaaccatg 1200 ccaaggtaaa
aaggtcaggg catggagaga gctatcggtt aaaaagtggc aggagagaca 1260
gcaactggct gcaagactca gaacttcttg gctgggcacg gtggctcacg cctgtaatcc
1320 cagcactctg ggaggccgag gcggggggat catggggtca ggagatcgag
accatcctgg 1380 ttaacacagt gaaaccccgt ctctactaaa aatacaaaaa
aattagccag gcatggtggc 1440 gggcacctgt agtcccagct actcaggagg
ctgaggcagg agaatggcgt gaacccggga 1500 ggcggagctt gcagtgagcc
aagatagcgc cactgcactc cagcctgggc aacagagcga 1560 gactccgtct
caaaaaaaaa aaaaaaaaaa aacttctttg gatcctgatc caaacaaact 1620
gccaagaaaa tgtttaggag ataatcatag agttttgaac aggagccaca tattagatga
1680 aatccaggaa ttattgttaa ttttatgagg tatcttaatg gtatcgtagt
gatgctacgc 1740 tctatcctag cccaggctgg agtgcagtgg cgcaatcaga
gttcactgca gttctgaact 1800 tcctggcctc aagcgatcct cccgtgtcag
cctctggaag tgctcggatt ataggcatga 1860 gccaccacac ccagcctgtt
gctttttttt tgtttgtttt aagaactctt atctctgaaa 1920 agtatgttcc
taaacattta ttgatttatt tacttattta tttttatttt tgagatggga 1980
tctcactctg ttgcccacgc tgaagtgcaa cgacgcagtc ttggctcact gcatcctctg
2040 cctcctggct caagcagtct ttccgcctca gcctcccgag tagctgggac
tacaggtgca 2100 gaccaccatg ctggctaatt tttgtatttt ttgtagagat
ggggttttgc catgttgtct 2160 aggctaggct ggtcttgaac acgtgagctc
aggccatccc ctcacttcag cctctcaaag 2220 tgctagaatt acaggcatga
gctggcttct aaacatttat gaatggaatg atggggtgtc 2280 tgggaggcag
gggaatagaa atgatgtaaa ctggacccca agttggcaag agtcagagct 2340
gggcgatgga tttgtggggt tcctcgtgtc cctcattagt tagtattcac tctcctttag
2400 tgcacgtgtg agattttcca tggtcaaaca gacaaatgct tgcactgaac
ctcccaggag 2460 aagcagagac agatggtgca agggccccag ggaagactta
cctttcactt aagataaatt 2520 tcccatcttt gaggctgggc agcttcctga
gggggttgat gtcaatgtat cctttgctgt 2580 ggtggtgacc tgggaggggc
agggaaggtc tgaggctgtg ggactccagg ggagagagaa 2640 ctgagactcc
cagagaccca aacgcctccc tctctatttt ctcaagaaga gggaactgag 2700
gcccggaggg acattgcgtc tcaccccagg tcacagggca aggcagttgc agaaccggac
2760 tgcgatcaga actgctggct cccagcctgc tccaccctag gtttggtgac
tcccgtgcct 2820 cctacctgtg tcccaggacc aggacgaccc ttttacccag
aagccggagg cctccagtgc 2880 ccacccccaa agctggatct gaaaacacag
cctttgaatc acctgaagcc ctgagggcct 2940 gggtcccatc cgcaatccca
tcgctctcac tctgtctcca ctttaaggaa gccaggccca 3000 gcacacagct
ggacatccaa agggaagctt ctcggacaca atcagggtca tcttaacagg 3060
gaacctgagg tgggggcagg aactgaaact cttcctggac cagccgcctc cagttggaaa
3120 catttctggg ggctccactc gcagcccgtt catttccaca gcttccctgt
ctcttcctct 3180 gtgttctaga ggcttctgct tttgcaggct gagcttttgg
agtccctctg tgctggggat 3240 ggagttggag cccacccctc tgaccctcac
tcagggttag tggagccctg agcctttctg 3300 aacactgggg aggatgggtg
tagacggact gtgcacttct gccccctttg ccaacctggt 3360 gggcaggtgc
tgagttcaca aggtcctaga atcccacaag gaagccaggg tgcctggtgg 3420
gagcccaggg agtcccagct actgttcctt cccccttctc ctcgaaaagc ctgttcatct
3480 gtggcgtggg gactgtcatt agtgagcact gactaaggta ggctggacaa
ggatgcagcc 3540 tacaagccgc gtggcatctt ttccttccct gtggacctct
ggggtgattc ccttgtctct 3600 gtctctgctc ctcagaaacg cccctatcag
gctgtgcgcg gtggctcacg cctgtaatcc 3660 cagcactctg gaggctgagg
tgggcagatc acttgaggtc aggagtttga gaccagcctg 3720 gccaacatgg
tgaaacccct gttaaaaata caaaaaatta gctgggcgtg gtggcatgca 3780
cctctaatcc cagctactcg ggaggctgag gcaggagacg cacttgaacc cagcagaggt
3840 tgcagtgagc cgagatagca ccaccgtact ccatgctggg caacagagcg
agactccatc 3900 aaaaaacaag aaaaaaagaa aagccgcaat ctgtgtgtcc
tgcctccccc caggaccagg 3960 cctgccaggc agcagtggga gttgaccttt
cagcagatcc acaaactgaa agttgaactg 4020 gatgtcatgc ttcttcgaga
agatgtagac ggcacggcag ggtgctgaca gcaggtccat 4080 gtagagctcc
agtgccatgt tgagacacat gccaggcccc acagccgcag ttggccagcc 4140
acagacctgg gcctatgtct ggccagagtc cctggccctg tgccctctcc gatctgggcc
4200 caggatcctg tgttccccag ggaaacctct tgtttccctt tgtgttgtca
taaggccagg 4260 aagcctgcaa ttctcacagc atcaaggatt ctaaggaggc
ccaggagtag gctggggaga 4320 ggcccgtggc aaaggtgtgg cagccgtgac
cctactctcc cccttccacg tgtgcctgtg 4380 ccccgtggtg ccacctcaca
gacaccagtc tgagaaggga ttatgcctgg gaattcccac 4440 ggctggattt
tcattgcaga acctgacgaa aggggctttg cagggtccag aatgaagagg 4500
aggcaatgag aattatccct ggaggattct agaagtagag gctgggagta tccacaggta
4560 aatcgagcct gaactatgac tagaaaggaa ttgggagaaa gagacacagg
tgaatcgagc 4620 ctgaactatg actagaaagg aactgggaga aagagacaca
ggaaactgtg agctttggga 4680 gcaatgggga caccaccacc aggaagtcag
ggggcactca gccggtgtgt gccacacaga 4740 ggagcctaga aacttcctgg
ccttggttgg ggctgcagtg gccagactgt gtacctggtg 4800 gccaaggaag
gtaactagag ccccacgtag aggactgagt gccactcact ctatgctgtg 4860
atctaatagg tctaggctga gaaatgggac tgaccccact tctggtgaca gagtaagcct
4920 ggagacaagc gaagagcatg cagtgtgttt attgcagaca gcagggtgca
gtggagtggg 4980 ctgcacccac tgcacctgct 5000 2 5000 DNA Homo sapiens
2 tgtggtttta attttgtttt tcatccttta ttgcaaatgt atactattga tttttgtgta
60 ttaatcttgt accctgcaac cttgctgaac ttattttatt ttattgagat
ggagtctcag 120 tcagtcaccc aggctggagt gcagtggctt gatcttggca
cactgcaacc tccacctcct 180 gggctctggt gattctcctg cctttgcctc
ctgagtagct gggactacag gcatgcgcca 240 ccacgcccgg ctaatttttg
tatttttagt agagacgggg tttcaccata ttggacaggc 300 tggtctcaaa
cttctgacct catgatctgc ccgccttggc ctcccaaagt gctgggatta 360
caggtgtgag ccaccgtgcc cagccttgaa cttgtttatt agctctaatg gtttttaagt
420 gagttaagaa tttctgtaat gtcatctgtg aatagagatg gttttacttc
tttctttctg 480 atctagattc caccttgttc ctttttcttg cctaattgcc
ctaataagta gaagtagtgt 540 gaatgaacat tcttgtcctg ttcctgatct
taggggggaa acgcttgatc tttcacaatg 600 gatgaagtat ggtgttagtt
ataggttttt catagatgct ttttgtcaag ttgaggaagt 660 tcctttctcg
tcttcatctg ctgagtaatt ttatcgtaaa aggatgttag gttttgtcaa 720
atgccttctg tgcattagga tgatcatgtg actttctatt aacatggtat gctacactga
780 ttgatttttg tatgttgaac tgcatttgta ctcctgggat aaatcctacc
tggtcaaggt 840 gtatgatcct tttaatatgc tgctggattt gatttgctaa
tattttgctg aggattttta 900 catctatatt tataaggata ttgctttgta
actttatttt cttgtaatgt ctttttctgt 960 ctttggtatc agagtaattc
tggcctcata gtatgagttg aaaaatattt cttcctgttt 1020 tatttttttg
gaagagtttg tgaaggcttt gtgttagttc ttcaagcttt tggttgaatt 1080
caccagggaa gccatctggt cctgggcttt tctttgtgga atttttaaaa ataattaata
1140 ttttaatctc tttatttgtt acaggcctat taaaatttta tctttcttct
taagtcagtt 1200 ttggtagatt gtgtgtttct aaaattttcc catttcatct
aggttgtcta aattgtccac 1260 atatagttat tcatagtatt tctaaacttt
tgaatttctc tatgaccaat gtgatgtctc 1320 cattttcttt ctttctggtt
tccatttcat tttttcattt ttgtttttgt ttgttttttg 1380 agataaggtt
cttctatgtg cccaggctgg agtgcagtgg tgcaatcata gctcggtgta 1440
accttgaact cttggactcg agtgatcctc ccacctcggc cacccagtta gctaagacta
1500 caggcttgca ccaccacacc aagctaattt ttttaaaaaa tatatttttt
agaaaaaagt 1560 ctcattgtgt tacccaggct gttatcaatc tcctggcctc
aagtgatcct tctgcctctg 1620 ctttccaaag tgctgggatt gcaggtgtga
gccattgcac ccagcctcca ctttctttct 1680 tggtgaataa tttgaccctt
atttttttca cttggctatt ctaagtaaag gtttgccaat 1740 tttgttgatc
tttgcaaaga tccaattttt ggttttattg attttattgc ttttctattt 1800
tctatttcat ttatctccat tctaatcttt attatttcct gttttctagt ggtgttgggt
1860 tgagtttgct cttatttttc tagttcctta aggtgtaaag ttaggttatt
gattcaagat 1920 cattcttctt taacttatgt gtttacagct ataaattttg
ctcttagaac tgcttctgct 1980 gcatcctata aatctgggca tgttgtgttt
tcatttttat ttgttcagga tactttttga 2040 tgtcccttgt gatttcttct
ttaacccatt tgttgtttaa gagtgtgctg tttaatttct 2100 atgtgcttgt
gagttttcta gttttccttc tgttcttgat ttttaacttt attccactgt 2160
gtccagagaa catactttgt gtgatttcaa tcatcttaaa tttgagactt aggaccaaat
2220 atatgctcta tactggagaa tattccatgt gctctttaga agaatgctta
ttctgctcct 2280 gtcgggtgga acgttctgtt catgtctagt gtatgcgatt
ggttaatcgt gcttttcaaa 2340 ttccctattt ccttgatggt cttctgtagt
ttttctatta ctgaaagtgg ggtattgaag 2400 tcaccaacta ttattactca
caatgtaacc atatttaacc ctttactttt aaaattcttt 2460 ttggaaactg
gaaggatctg taaccaccac ccactcccac atcagaccct atgcatatgc 2520
caactgtctg ctgctcagca gctgtgatgg ttgttccagt tctttattag gcaaagaaca
2580 gttgtttttt tgtttttttg tttttttttt tttaacattt cctttaagga
aggtggctca 2640 gattgctaag ccagccaggc cctgcgggac aggctgcgcc
tagggtcacc tgctcttctt 2700 cagcaactca gaaatattct ccttgaccat
tgaatccaat gttgaaaagt cccagtcggc 2760 caactgcatt agtcgatcat
gggcctccct aaagaggcca gagccaatat tcagctccac 2820 ctgcatacgc
cactcagcta gcttggagct gttgaggaag acattatagt tggctgccat 2880
gggctgtaga tagacgaaga caaagacgtg gtcagcctgg ggaccagccc cacctgggtc
2940 ctctcccaca gcctcaggcc cacgtcccta acatctacct atgcaaagaa
ttaggctgct 3000 gacccccaag gcctgagcaa ggggtcacag actatgtaat
agatgtaggt ggggatggag 3060 gatccagaat ccctcagagt cctcagtcac
tggtcttctt gctccaagcc ttctgaacca 3120 tgctgagagg gctcctggcc
caggcacttc ccacttctcc agcctgcgac ctcgcatgaa 3180 tcctgccttc
ttccagggaa accccttatc ccaggtgtgt atatgtggat gagggagaga 3240
actcaggttt tttcctccat gtttagcctc ccactgtgat caagctcagg ggctaggatg
3300 ggagacctgg cgggcagtct accctgcagt ttcgctggct tactaagagt
ttggtttgca 3360 cccaagatct ttgggaagcc caagaatggg tgtgtgtggg
tgaaatgtaa ggggtgggga 3420 cgaagcatat ggctgaaccc ttggggcagg
ccagaatgat ttttcctggt gctggtctgc 3480 cctgcaaaca gaccaaggag
actaattttc atatcagcca gagatctctg ggataaggaa 3540 aagaatactg
cattttctgg tcaatccacc aggaccccag gtccctcctc tcgggcatat 3600
ctctggctga tatgcaaatt agtctctttg gtcagtgtgc agtgccctag ctggtgtgca
3660 ggatggccag ttgagaccct ggccagtgtc ttgacaagca gaactggtca
ccctcccctg 3720 catgtagagg ccacataaat gccccacact caggtgtgcc
tccaaatgca cagtggatgc 3780 ccctcagacc cagccacgag agctgtcctc
cagagctgtc tgtctggagc tctgggaaac 3840 aggcagggcc agaaggacac
ccaggaagcc agtgaacatt tcctggagag tccagcaaga 3900 ggaggaggta
tctgggatgc tggtggattg agcaggaaat gcagtgttct tctctatccc 3960
aggctcaccc tccgggtcct cccacaccga agaatctttg tcaagtgtgg agaactgtga
4020 tccttcctga ttcataacat tctgtgcttc ctgttgcccc gattgagtcc
aggcccccag 4080 gcctggttcc cgcagccccc atggcagctc tgcctgcctt
tcccgcctca ccagcctatc 4140 ctcaagtgat ggccccattg gtcacagagg
agtcctacct ctgcccaggg tctaaccctc 4200 ctccaatcca ctccacacct
gcatcatctc caccacggcc accaggtcag ccagtgagat 4260 ttggttcccg
gtgatgaaca tcttatcctg cagaaaatac tcctcaaaga gctgcaggct 4320
gttcttcacc tcttccactg catgctccat cttctcagct gaaacttcct cccctgttat
4380 ctttgggatc agcaactggc cagggttggg aagaggaggg aagaggaggc
tgcactccag 4440 ggccacctgc cctgccaggt ctctgtactc ttgtctgctg
gatagatatt gaacacttcc 4500 caggatataa agcagtttca cctcttttag
cagttctgat tggtggaagt tgctgggaac 4560 catgtgttca caaggatttg
gggagctcag caggcataag tcctgtgatt gattagtgat 4620 gtctgtcaca
ggcatagaat tcaaagtaga gacacatgta ctggttattt gtcatcttct 4680
aattttctat aggccatact cttttttgtt tttgtttttt gagatggagt ctcactctgt
4740 cgcccaggct ggagtgcagt ggcacaatct tggctcactg ccaactccaa
ctcctgggtt 4800 caagcaattg tcctgcctca gcgtcctgag tagctgggat
tacaggtgcc catcaccaca 4860 cccagctaat aattttgtat ttttagtaga
gatggtgttt cacaatgttg gcaaggtagg 4920 tcttgaactc ctgacctcaa
gtgatctgcc cgcctcggcc tcccaaagta ctgggattac 4980 agttgtgagc
caccgtaccc 5000
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